Scanning Electron Microscopy - SEM

Electron Microscope
• Electron Microscopes are instruments that use a beam of highly
energetic electrons to examine objects on a very fine scale.
• This examination can yield information about the:
• Topography
• Morphology
• Composition
• Crystallographic information

Mainly 2 types:
• Transmission Electron Microscope (TEM) - allows one the study of the
inner structures.
• Scanning Electron Microscope (SEM) - used to visualize the surface of
objects

Properties of electron
• Electron are used as a source of illumination
• They are negatively charged subatomic particles
• When the atoms of metal are excited by sufficient energy in
the form of heat, the electron leave their orbit, fly off from
space & are lost in atoms
• Metal tungsten is commonly used as a source of electron

• The electron are readily absorbed & scattered by different form of
matter
• So a beam of electron -> produced & sustained only in high vacuum
• Electron are like light waves-> So used in image formation
• Electron interact with the atoms of the biological specimens to form
the image

Electron beam
(A)
Transmitted
electron
(B)
Inelastically scattered
electrons
(C)
Elastically scattered
electrons
(D)
Back-scattered electrons
(E)
Secondary electrons
X-rays
Visible light

1. Transmitted electrons (A) of the beam passes straight through the
specimen on to the screen
2. Some electron (B) of the beam lose a bit of their energy while passing
through the specimen & get deflected a little from their original axis of
the beam  inelastically scattered electrons
3. Some electron (c) interact with atoms of specimen & get elastically
scattered without losing energy. Electron deviate widely
4. Some electron (D) get backscattered instead of getting transmitted
through the specimen
5. In some cases the electrons get absorbed by the atoms of the specimen
& instead low energy electron (E) are emitted. These electron are
termed secondary electron. These are very useful for forming the image
in the SEM
6. Some atom emit x-ray & light energy

Scanning Electron
Microscope (SEM)

Principle
• The basic principle is that a beam of electrons is
generated by a suitable source, typically a
tungsten filament or a field emission gun.
• The electron beam is accelerated through a high
voltage (e.g.: 20 kV) and pass through a system
of apertures and electromagnetic lenses to
produce a thin beam of electrons.
• Then the beam scans the surface of the
specimen Electrons are emitted from the
specimen by the action of the scanning beam
and collected by a suitably-positioned detector.

 Topography:
The surface features of an object or "how it looks", its texture;
direct relation between these features and materials properties
 Morphology:
The shape and size of the particles making up the object; direct
relation between these structures and materials properties
 Composition:
The elements and compounds that the object is composed of
and the relative amounts of them; direct relationship between
composition and materials properties
 Crystallographic Information:
How the atoms are arranged in the object; direct relation between these
arrangements and material properties.
Characteristic Information: SEM

Construction
Scanning Electron Microscope’s basic components are as following…
1. Electron gun (Filament)
2. Condenser lenses
3. Objective Aperture
4. Scan coils
5. Chamber (specimen test)
6. Detectors
7. Computer hardware and software

Electron Guns
Electron guns are typically one of TWO types.
1) Thermionic guns
2) Field emission guns
• Thermionic guns: Which are the most common
type, apply thermal energy to a filament to
coax electrons away from the gun and toward
the specimen under examination.
• Usually made of tungsten, which has a high
melting point

Field emission guns:
• create a strong electrical field to pull electrons away from the
atoms they‘re associated with.
• Electron guns are located either at the very top or at the very bottom
of an SEM and fire a beam of electrons at the object under
examination.
• These electrons don't naturally go where they need to, however,
which gets us to the next component of SEMs.

Anode [Hitachi S2300]
ELECTRON SOURCES

Condenser Lenses
• Just like optical microscopes, SEMs use Condenser lenses to produce
clear and detailed images.
• The Condenser lenses in these devices, however, work differently.
• For one thing, they aren't made of glass.
• Instead, the Condenser lenses are made of magnets capable of
bending the path of electrons.
• By doing so, the Condenser lenses focus and control the electron
beam, ensuring that the electrons end up precisely where they need
to go.

Objective Aperture
• The objective aperture arm fits above the objective lens in the SEM.
It is a metal rod that holds a thin plate of metal containing four holes.
Over this fits a much thinner rectangle of metal with holes (apertures)
of different sizes. By moving the arm in and out different sized holes
can be put into the beam path.
• An aperture holder: this arm holds a thin metal strip with different
sized holes that line up with the larger holes. The metal strip is called
an Aperture strip.
• The aperture stops electrons that are off-axis or off-energy from
progressing down the column. It can also narrow the beam below the
aperture, depending on the size of the hole selected.

Scan Coils
• The scanning coils consist of two solenoids oriented in such a way as
to create two magnetic fields perpendicular to each other.
• Varying the current in one solenoid causes the electrons to move left
to right.
• Varying the current in the other solenoid forces these electrons to
move at right angles to this direction (left to right) and downwards.

Chamber (Specimen Test)
• The sample chamber of an SEM is where researchers place the specimen
that they are examining.
• Because the specimen must be kept extremely still for the microscope to
produce clear images, the sample chamber must be very sturdy and
insulated from vibration.
• In fact, SEMs are so sensitive to vibrations that they're often installed on
the ground floor of a building.
• The sample chambers of an SEM do more than keep a specimen still.
• They also manipulate the specimen, placing it at different angles and
moving it so that researchers don't have to constantly remount the object
to take different images.

Detectors
• SEM's various types of detectors as the eyes of the microscope.
• These devices detect the various ways that the electron beam
interacts with the sample object.
• For instance, Everhart-Thornley detectors register secondary
electrons, which are electrons dislodged from the outer surface of a
specimen. These detectors are capable of producing the most
detailed images of an object's surface.
• Other detectors, such as backscattered electron detectors and X-
ray detectors, can tell researchers about the composition of a
substance.

Detectors
Secondary electron detector:
(Everhart-Thornley)
Backscattered electron
detector:
(Solid-State Detector)

Vacuum Chamber
• SEMs require a vacuum to operate.
• Without a vacuum, the electron beam generated by the electron gun
would encounter constant interference from air particles in the
atmosphere.
• Not only would these particles block the path of the electron beam,
they would also be knocked out of the air and onto the specimen,
which would distort the surface of the specimen.

How do we get an image?
156 electrons!
Image
Detector
Electron gun
288 electrons!

• The electron gun produces an electron beam when tungsten wire is heated by current.
• This beam is accelerated by the anode.
• The beam travels through electromagnetic fields and lenses, which focus the beam down
toward the sample.
• A mechanism of deflection coils enables to guide the beam so that it scans the surface of
the sample in a rectangular frame.
• When the beam touches the surface of the sample, it produces:
– Secondary electrons (SE)
– Back scattered electrons (BSE)
– X - Rays...
• The emitted SE is collected by SED and convert it into signal that is sent to a screen which
produces final image.
• Additional detectors collect these X-rays, BSE and produce corresponding images.

• A focused electron beam (2-10 keV) scans on the surface, several types of
signals are produced and detected as a function of position on the
surface.
• Different type signal gives different information: a. Secondary electrons:
surface structure.
b. Backscattered electrons: surface structure and average elemental
information.
c. X-rays and Auger electrons: elemental composition with different
thickness-sensitivity.

Electron beam-sample interactions
• The incident electron beam is scattered in the sample, both
elastically and inelastically
• This gives rise to various signals that we can detect (more on
that on next slide)
• Interaction volume increases with increasing acceleration
voltage and decreases with increasing atomic number

Secondary electrons (SE)
• Generated from the collision
between the incoming electrons and
the loosely bonded outer electrons
• Low energy electrons (~10-50 eV)
• Only SE generated close to surface
escape (topographic information is
obtained)
• Number of SE is greater than the
number of incoming electrons

Backscattered electrons (BSE)
• A fraction of the incident electrons is
retarded by the electro-magnetic field of
the nucleus and if the scattering angle is
greater than 180 ° the electron can escape
from the surface
• High energy electrons (elastic scattering)
• Fewer BSE than SE

Choose correct detector- topography example
Fracture surface in iron
backscattered electrons secondary electrons

• A secondary electron detector attracts the scattered electrons and,
depending on the number of electrons that reach the detector,
registers different levels of brightness on a monitor.

 A low atomic weight
area of the sample will
not emit as many
backscattered
electrons as a high
atomic weight area of
the sample.
 In reality, the image
is mapping out the
density of the sample
surface.

X-rays
• Photons not electrons
• Each element has a
fingerprint X-ray signal
• Poorer spatial resolution than
BSE and SE
• Relatively few X-ray signals
are emitted and the detector
is inefficient
 relatively long signal
collecting times are needed

SCANNING ELECTRON MICROSCOPIC IMAGE OF
THE TONGUE

Scanning electron micrographs of the early
human embryo

• This form of image processing is only in gray scale which is why SEM
images are always in black and white.
• These images can be colorized through the use of feature-detection
software, or simply by hand editing using a hand graphic editor.
• This is usually for aesthetic effects, for clarifying structure, or for
adding a realistic effect to the sample

Pollen and Stamens Wool fibers

Specimen
Can examine
fracture surfaces
electronic devices
fibers
coatings
particles
etc.

Specimen
What comes from specimen?
Backscattered electrons
Secondary electrons
Fluorescent X-rays
high energy
compositional contrast
low energy
topographic contrastcomposition - EDS
Brightness of regions in image increases as
atomic number increases
(less penetration gives more
backscattered electrons)

Applications
• SEMs have a variety of applications in a number of scientific and industry-
related fields, especially where characterizations of solid materials is
beneficial.
• In addition to topographical, morphological and compositional
information, a Scanning Electron Microscope can detect and analyze
surface fractures, provide information in microstructures, examine surface
contaminations, reveal spatial variations in chemical compositions, provide
qualitative chemical analyses and identify crystalline structures.
• In addition, SEMs have practical industrial and technological applications
such as semiconductor inspection, production line of miniscule products
and assembly of microchips for computers.
• SEMs can be as essential research tool in fields such as life science, biology,
gemology, medical and forensic science, metallurgy.

Advantages
• Advantages of a Scanning Electron Microscope include its wide-array of applications, the
detailed three-dimensional and topographical imaging and the versatile information
garnered from different detectors.
• SEMs are also easy to operate with the proper training and advances in computer
technology and associated software make operation user-friendly.
• This instrument works fast, often completing SEI, BSE and EDS analyses in less than five
minutes. In addition, the technological advances in modern SEMs allow for the
generation of data in digital form.
• Although all samples must be prepared before placed in the vacuum chamber, most SEM
samples require minimal preparation actions.

Disadvantages
• The disadvantages of a Scanning Electron Microscope start with the size and cost.
• SEMs are expensive, large and must be housed in an area free of any possible electric,
magnetic or vibration interference.
• Maintenance involves keeping a steady voltage, currents to electromagnetic coils and
circulation of cool water.
• Special training is required to operate an SEM as well as prepare samples.
• SEMs are limited to solid, inorganic samples small enough to fit inside the vacuum chamber
that can handle moderate vacuum pressure.
• The sample chamber is designed to prevent any electrical and magnetic interference, which
should eliminate the chance of radiation escaping the chamber. Even though the risk is
minimal, SEM operators and researchers are advised to observe safety precautions.

1. Cleaning the surface of the specimen
2. Stabilizing the specimen
3. Rinsing the specimen
4. Dehydrating the specimen
5. Drying the specimen
6. Mounting the specimen
7. Coating the specimen
SEM SAMPLE PREPARATION

SEM SAMPLE PREPARATION
Cleaning the surface of the specimen
 Very important
 Surface contains many unwanted deposits, such as dust, mud, soil etc
depending upon the source of the sample/specimen.

Stabilizing the specimen
 Hard, dry materials such as wood, bone, feathers, dried insects,
or shells can be examined with little further treatment, but living
cells and tissues and whole, soft-bodied organisms usually
require chemical fixation to preserve and stabilize their
structure.
 Stabilization is typically done with fixatives.

Fixation
 performed by incubation in a solution of a buffered chemical
fixative, such as glutaraldehyde, sometimes in combination
with formaldehyde and other fixatives.
Fixatives that can be used are:-
1. Aldehydes.
2. Osmium tetroxide.
3. Tanic acid.
4. Thiocarbohydrazides.

Rinsing the specimen
 Sample must be rinsed -- remove excessive fixatives.
Dehydrating the specimen
 Water must be removed
 Air-drying causes collapse and shrinkage, this is commonly achieved by
replacement of water in the cells with organic solvents such
as ethanol or acetone.
 Dehydration -- performed with a graded series of ethanol or acetone.

Drying the specimen
 Specimen should be completely dry
 Otherwise the sample will be destroyed
Mounting the specimen
 Specimen has to be mounted on the holder
 Mounted rigidly on a specimen holder called a specimen stub
 Dry specimen -- mounted on a specimen stub using an adhesive such as epoxy
resin or electrically conductive double-sided adhesive tape.

• To increase the conductivity of the specimen and to prevent the
high voltage charge on the specimen
• Coated with thin layer i.e., 20nm-30nm of conductive metal.
• All metals are conductive and require no preparation before being
used.
Coating the specimen

Coating the specimen
Non-metals need to be made conductive
Done by using a device called a "sputter coater”
Conductive materials
Gold
Gold-palladium Alloy
Platinum
Osmium
Iridium
Tungsten
Chromium
Graphite

BIOLOGICAL APPLICATIONS OF
SEM
• Virology - for investigations of virus structure
• Cryo-electron microscopy – Images can be made of the surface of frozen
materials.
• 3D tissue imaging -
– Helps to know how cells are organized in a 3D network
– Their organization determines how cells can interact.
• Forensics - SEM reveals the presence of materials on evidences that is otherwise
undetectable
• SEM renders detailed 3-D images
– extremely small microorganisms
– anatomical pictures of insect, worm, spore, or other organic structures

Advantages
• It gives detailed 3D and topographical imaging and the versatile information
garnered from different detectors.
• This instrument works very fast.
• Modern SEMs allow for the generation of data in digital form.
• Most SEM samples require minimal preparation actions.
Disadvantages
• SEMs are expensive and large.
• Special training is required to operate an SEM.
• The preparation of samples can result in artifacts.
• SEMs are limited to solid samples.
• SEMs carry a small risk of radiation exposure associated with the electrons that
scatter from beneath the sample surface.
ADVANTAGES & DISADVANTAGES OF SEM

Scanning Electron Microscopy - SEM

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